Journal of Advanced Laboratory Research in Biology E-ISSN: 0976-7614 Volume 5, Issue 3, July 2014 PP 35-40 https://e-journal.sospublication.co.in

Research Article

Control of solanacearum Infection in , Brinjal and Capsicum by antibiotic sensitivity test

Rupa Verma1*, Abhijit Dutta2, Ashok Kumar Choudhary1, Sudarshan Maurya3

1Department of Botany, Ranchi University, Ranchi - 834008, Jharkhand, India. 2Department of Zoology, Ranchi University, Ranchi - 834008, Jharkhand, India. 3ICAR-Research Complex for Eastern Region, Research Center, Ranchi - 834001, India.

Abstract: Ralstonia solanacearum is one of the important dreaded soil’s borne bacterial phytopathogen which causes enormous losses in the crop plants in tropical, subtropical and temperate region of the world. In India, the disease is highly prevalent and active throughout the year where the soil is acidic in the Eastern Plateau and Hill Region. Once the disease is established in the field, it cannot be controlled by chemical means. Keeping these in view, screening of potential antibiotics for the management of R. solanacearum was done. In the present study, three strains of R. solanacearum were isolated from Brinjal, Tomato and Capsicum. Against these three strains of R. solanacearum, four antibiotics were screened through food poison techniques viz. Kasugamycin, Streptomycin, Ceftriaxone and Gentamicin. The different strains of R. solanacearum and antibiotic sensitivity showed varied response. Among the screened antibiotics, Gentamicin showed strong antibacterial efficacy which inhibited 100% Colony-forming Unit (cfu) at very low concentration (1 ppm) followed by Ceftriaxone which inhibited >50% cfu at 1 ppm against all three strains. However, Streptomycin also showed antibacterial efficacy and inhibited >50% cfu at 3 ppm, but Kasugamycin was found less antibacterial as compared to other tested antibiotics and inhibited >50% cfu at 4 ppm.

Keywords: Bacterial wilt, Antibiotics, Ralstonia solanacearum, antibacterial.

1. Introduction parenchyma and enters the vessel and spreads up into the stems and leaves. The races of Ralstonia Bacterial wilt is a major disease in solanaceous solanacearum identified by pathogenicity test on wide crop caused by Ralstonia solanacearum1. The disease host range6. The species of this bacterium were affects a wide range of economically important crops classified into five races on the basis of different host such as Tomato, , , Chilli and non- range7 and six biovars, according to the ability to solanaceous crop such as Banana and Groundnut in oxidize disaccharides and three sugar alcohols8,9. India2. The causative agent is the bacterial During the following year, five biovars (based on Ralstonia solanacearum, which was formerly known as carbon utilization pattern) and five pathogenic races solanacearum and is the causative agent were identified. Race1 occurs in the lowland tropics of bacterial wilt of solanaceous crop3. It mainly attacks and warm temperate lands10. It colonizes the xylem, Potato, Tomato, Geranium, Eggplant, Capsicum and causing bacterial wilt in a very wide range of host some solanaceous weeds like Solanum nigrum and plants11. The disease is called southern blight, R. Solanum dulcamara4, 5. solanaceous wilt and other common names where it It is an important soil borne, devastating bacterial occurs. The virulent and avirulent isolates of this phytopathogens, which are aerobic in nature, non-spore bacterium were identified by Kelman’s Tetrazolium former, Gram-negative and is motile by a polar chloride (TZC) agar test. In this test, virulent isolates flagella tuft. This bacterium normally invades produce pink or light red colour colonies with through wounds and colonizes the cortex and vascular

*Corresponding author: E-mail: [email protected]. Control of Ralstonia solanacearum in Tomato, Brinjal and Capsicum Verma et al characteristic red colour center with whitish margin areas of Ranchi, Jharkhand, India. The typical and avirulent produce off white margin8. symptoms of bacterial wilted plants were observed. It was reported that avirulent type could be easily Infected leaves of the plants by Ralstonia differentiated by the pigmentation from the virulent solanacearum turned yellow and remain wilted after a types12. In India, a study showed 10 to 100% incidence time. The area between leaf veins died and becomes of bacterial wilt in summer13. The soil born vascular brown. Usually, the main stem of the affected plants pathogen is widely distributed in tropical, subtropical remained upright even though all the leaves may climates and effects usually broad range of crops become wilted and dead. Stem streaming methods were including monocots and diacot plants8, 14. Infected soil a valuable diagnostic tool for quick detection of and surface water, including irrigation water are the bacterial wilt in the field21. A thin thread of ooze was primary sources of the inoculum. The pathogen infects seen when an infected stem cuts across and cut ends roots of susceptible plants, usually through wounds15. held together for a few seconds in test tubes containing Colonization by the bacterium within the xylem sterilized distilled water. Slimy sticky ooze forms tan- prevents water movement into the upper portion of white to brown beads. Internal symptoms were also plant tissues12. As the disease develops, all leaves may obtained, a dark brown to black streaking in the wilt quickly and dessiccate although they remain vascular tissues was revealed. All three strains were green16. This bacterium was characterized into biovars isolated from wilted plants by streaking a loop full on the basis of oxidation of carbohydrate. Biovars of R. flowing ooze containing onto sterile TTC solanacearum were differentiated according to their media. Turbid bacterial suspensions were obtained ability to oxidize disaccharides and hexose alcohol8. when those stems were cut and placed in test tubes Aqueous extracts of some plants having medicinal filled with sterilized distilled water. Bacterial colonies properties did not have inhibitory effects on Ralstonia were obtained after incubation of 24-36 hrs. Red solanacearum17. The most commonly used chemical colonies were isolated. treatment has been fumigation of contaminated soil/portion of the farm with methyl bromide18. This is very expensive and tedious exercise and cannot be used on large areas. The other product used at field level is sodium hypochlorite. It is appropriate for spot treatment of the holes left behind after rogueing of the wilting plants and for general field sanitation19. Its use is expensive and tedious. Hence, is the need to study antibacterial agent other than solvent extract to repress the growth of this bacterium. In the present study, four antibiotics have been taken, namely Kasugamycin, Ambistryn, Ceftriaxone and Gentamicin. With an aim to develop effective antibacterial agent without any residual effect, the present study was conducted to analyze the in-vitro antibacterial potential of certain antibiotics Fig. 1(a). Wilt in Capsicum. against three different isolates of Ralstonia solanacearum. The antibacterial activities of antibiotics were assayed by using Food Poison techniques. Four above antibiotics were used against three isolates of Ralstonia solanacearum. These compounds, which include aminoglycosides, tetracyclines and macrolides, interfere with essential steps of protein synthesis. Most antibiotics interact with ribosomal RNA20. The ribosome is the target of many important antibiotics, inhibiting the protein synthesis of the bacterium and thereby ceasing its growth and propagation.

2. Materials and Methods

2.1 Field survey, Identification and Sample collection A field survey was conducted to determine the Fig. 1(b). Wilt in Tomato. prevalence of bacterial wilt in solanaceous crops such as Brinjal, Tomato and Capsicum, growing in different

J. Adv. Lab. Res. Biol. 36 Control of Ralstonia solanacearum in Tomato, Brinjal and Capsicum Verma et al

Gentamicin, whose chemical structures are shown in Fig. 4a-4d.

Fig. 1(c). Wilt in Brinjal.

Fig. 4(a). Structure of Kasugamycin.

Fig. 2. Flowing ooze containing bacteria. Fig. 4(b). Structure of Streptomycin.

2.2 Purification For further purification bacterium were inoculated in nutrient agar media by streak plate technique. In these media, two types of isolates were revealed. On the plates of Brinjal and Capsicum, isolates were whitish in color. Tomato isolates were golden yellow in color. For further use, bacterial isolate were stored in King’s B media. Finally, a pure culture of all these three isolates was obtained. Fig. 4(c). Structure of Ceftriaxone.

Fig. 4(d). Structure of Gentamicin.

Three replicates of each of the three isolates were tested against these drugs and four sets of experiment were done. For each set 45 Petri plates and 18 test tubes were taken, which were sterilized in hot air oven (160ºC for 1 hour). 1 litre Nutrient Agar was prepared Fig. 3. Bacterial isolates in TTC medium. and sterilized in the autoclave (121ºC at 15 psi for 20 min.) for each set of experiment. A stock solution of 2.3 Antibiotic sensitivity test antibiotics was prepared. Before pouring different An antibiotic sensitivity test of Food Poison concentration of antibiotics (1 ppm, 2 ppm, 3 ppm, 4 Technique was performed. Three isolates of the ppm) was added into the sterilized media (1 litre bacterium from Tomato, Brinjal and Capsicum were medium was divided into 5 flasks containing 100ml selected to test the antibacterial activity of four drugs, each, before sterilization). Bacterial suspension of all namely Kasugamycin, Ambistryn, Ceftriaxone, and three isolates was prepared by serial dilution up to 10-6

J. Adv. Lab. Res. Biol. 37 Control of Ralstonia solanacearum in Tomato, Brinjal and Capsicum Verma et al

and was inoculated by the spread plate method. After and interfere with the protein synthesis. These inoculation, these plates were incubated for 24 hours. antibiotics are acting on this bacterium due to its Percentage inhibition was calculated. bactericidal activity against gram-negative pathogen. Streptomycins usefulness has decreased greatly due to 3. Results and Discussion widespread drug resistance23. Streptomycin showed inhibition up to 50% CFU at 3 ppm, but Gentamicin The different strains of R. solanacearum and showed 100% CFU inhibition at 1 ppm. Ceftriaxone is antibiotic sensitivity showed varied response. Among a sterile, semisynthetic, broad spectrum cephalosporin the screened antibiotics, Gentamicin showed strong antibiotic. The bactericidal activity of Ceftriaxone antibacterial efficacy which inhibited 100% colony results from inhibition of cell wall synthesis. forming unit (cfu) at very low concentration (1 ppm) Cephalosporin also inhibits the transpeptidase reaction followed by Ceftriaxone which inhibited >50% cfu at 1 during peptidoglycan synthesis23. This has been ppm against all three strains. However, Streptomycin statistically confirmed. also showed antibacterial efficacy and inhibited >50% cfu at 3 ppm, but Kasugamycin was found less 3.1 Statistical Analysis antibacterial as compared to other tested antibiotics and The above findings were statistically analyzed inhibited >50% cfu at 4 ppm (Table-1). and the summary statistics for each of the selected The reason for bacterial growth inhibition, data variables shows measures of central tendency, according to Barbera et al., (2006)22 is due to the fact measures of variability, and measures of shape (Table- that Kasugamycin (Ksg) binds within the messenger 2). The standardized skewness, which is used to RNA channel of the 30S subunit. Ksg is also produced determine whether the sample came from a normal by the bacterium Streptomyces kasugaensis and has distribution. None of the values were found to be been used clinically in the treatment of Pseudomonas outside the range of -2 to +2 indicating that the values aeruginosa infections22. The drug Ambistryn contains outside the expected range were found to be none. This streptomycin. It is aminoglycosides and are synthesizes clearly shows that the experiments followed a normal by different species of the genus Streptomyces whereas, distribution as indicated by summary statistics of the Gentamicin comes from Micromonospora purpurea. three plant species selected. Aminoglycosides bind to the 30S ribosomal subunits

-6 Table 1. Antimicrobial efficacy of different antibiotics against three isolates of bacteria bacterial colonies (nx10 ) % inhibition.

% Inhibition Strains Conc. Antibiotics Kasugamycin Ambistryn Ceftriaxone Gentamicin 1ppm 30.96 39.63 80.79 43.57 2ppm 45.71 45 100 55.1 Tomato 3ppm 52.39 61.27 100 100 4ppm 63.35 75.23 100 100 1ppm 28.16 42.72 92.46 100 2ppm 36.3 86.36 100 100 Brinjal 3ppm 42 100 100 100 4ppm 56.73 100 100 100 1ppm 18.29 45.87 52.26 100 2ppm 33.48 68.01 70.4 100 Capsicum 3ppm 48.53 86.1 91.44 100 4ppm 56.29 99.38 94.4 100

Table 2. Summary statistics.

Kasugamycin Ambistryn Ceftriaxone Gentamicin Tomato Brinjal Capsicum Tomato Brinjal Capsicum Tomato Brinjal Capsicum Tomato Brinjal Capsicum Standard 16.171 12.045 16.8231 9.605 27.139 23.2014 29.628 3.77 19.7217 16.171 0 0 Deviation Coeff. of 29.25% 29.52% 29.52% 10.09% 32.99% 32.99% 39.68% 3.84% 3.84% 29.25% 0% 0% variation Stand. 0.4158 0.6034 -0.38782 -1.633 -1.3792 -0.3672 -0.1057 -1.633 -0.5385 0.4158 0 0 Skewness

J. Adv. Lab. Res. Biol. 38 Control of Ralstonia solanacearum in Tomato, Brinjal and Capsicum Verma et al

Fig. 5. Treatment of bacterial culture with Ceftriaxone.

3.2 ANOVA Result Gentamicin showed 100% efficacy at 1 ppm The values obtained were subjected to ANOVA concentration, hence can be effectively used alone for analysis (Table-3), which indicates that since the P- the treatment of the bacterial wilt by R. solanacearum. value of the F-test is less than 0.05, there is a Other antibiotics, used in the present study like statistically significant difference between the means of Ceftriaxone and Ambistryn, showed 50% efficacy at 1 the 4 variables at the 95.0% confidence level in all three ppm and 3 ppm, respectively, while Kasugamycin experimental plants and the antimicrobial efficacy is showed less effective in controlling the disease. sound in all four antibiotics. Hence, it can be concluded that Gentamicin at the concentration of 1 ppm can be successfully used to Table 3. ANOVA result. curb the menace of Ralstonia solanacearum infection and can be a boon to the farmers and can be used Tomato Brinjal Capsicum under field management; while other antibiotics can be ANOVA F-Ratio P-Value F-Ratio P-Value F-Ratio P-Value used in combination for effective management of the (Between 3.89 0.0373 13.51 0.0004 8.35 0.0029 disease. groups) Acknowledgment 4. Conclusion

We extend our sincere thanks to the Dr. Shivendu After statistical analysis, it is evident that since all Kumar, Director, ICAR-Research Complex for Eastern the three experimental plants showed 95.05% Region, Research Center, Ranchi, India, for kindly confidence level variables of antibiotics, the results are providing the necessary laboratory facilities to carry statistically significant. Among all the four antibiotics, out the research.

J. Adv. Lab. Res. Biol. 39 Control of Ralstonia solanacearum in Tomato, Brinjal and Capsicum Verma et al

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